P. gingivalis, an oral pathogen linked to adult periodontitis, encounters many different environmental conditions during colonization, biofilm formation and host cell invasion resulting in complex patterns of gene regulation. However, the mechanisms of gene regulation in response to changing environment remain unclear. Regulation of gene expression, in response to a specific condition, by small non-coding regulatory RNAs (sRNA) is an emerging paradigm in biology. In bacteria, sRNAs regulate gene expression via a number of mechanisms, including transcription, translation and mRNA stability. This mechanism is widespread in bacteria and is commonly employed by pathogenic bacteria to control virulence. However, the role of sRNAs in P. gingivalis has not yet been explored. We hypothesize that P. gingivalis expresses sRNAs to regulate gene transcription and virulence. To test this hypothesis we probed high-density whole-genome tiled oligonucleotide arrays with cDNA synthesized directly from an RNA fraction of 200 nt or less and identified thirty seven sRNAs regulated by hemin availability, a condition under which many virulence genes are regulated. As our overall goal is to understand the role and mechanisms of sRNA regulation in P. gingivalis, the purpose of this proposal is to characterize the sRNAs identified by array and to explore the biological role of those most highly regulated by hemin-limiting and standard hemin growth conditions. Aim 1 will characterize the sRNAs using a combination of Northern Blot analysis and RACE (rapid amplification of cDNA ends). The mode of sRNA regulation is at the post transcriptional level, or more rarely, by regulation of a protein which in turn regulates transcription. The result of any of these regulatory mechanisms would be a change in protein expression, therefor we propose in Aim 2 to define the biological role of the sRNAs by a comparative proteomics approach using multidimensional protein identification technology (MudPIT). These studies will uncover the biological roles of novel sRNAs as well as provide insight into a presently unexplored mechanism of gene regulation in P. gingivalis. PUBLIC HEALTH RELEVANCE: This proposal aims to identify and characterize the role of small regulatory RNA molecules in regulation of the pathogenesis of Porphyromonas gingivalis. This bacterium is an oral pathogen of adult periodontitis and is implicated in other more systemic diseases such as cardiovascular disease and pre-term delivery of low birth weight infants. Our work may ultimately identify unique molecules against which to design novel therapeutic treatments.